Project Summary A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales?including those involved in cancer progression and migration?requires new imaging tools. The long-term goal of our work is to develop general strategies for macroscopic, multi-cell tracking in living organisms. The objective of this application is to engineer novel bioluminescent tools for sensitive, multi-cellular imaging in vivo. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging use the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. Over the previous granting period, we demonstrated that the substrate-binding interface of firefly luciferase can be re- engineered to generate panels of mutant enzymes that accept chemically distinct luciferins. When mutants and analogs are mixed together, light emission is produced only when complementary enzyme-substrate partners interact. Several pairs of orthogonal enzymes and substrates were identified, but they remain weak emitters and not suitable for sensitive imaging in vivo. Our central hypothesis is that improved orthogonal imaging tools can be generated using a combination of rational design and screening. Guided by strong preliminary data, our work will encompass the following specific aims: 1) Identify the molecular determinants of orthogonality for lead pair optimization; 2) Generate orthogonal probes with improved tissue penetrance; and 3) Image tumor heterogeneity with expanded orthogonal toolsets. Under the first aim, we will use crystallography and deep-sequencing analyses to examine enzyme-substrate interactions responsible for orthogonality. These insights will be used to optimize existing orthogonal luciferase-luciferin pairs. In the second aim, we will prepare bioluminescent tools with red-shifted emission spectra. These tools will provide more sensitive imaging in vivo. In the third aim, the enzyme-substrate pairs will be used to address the roles of distinct cellular subsets in heterogeneous tumor models. Methods to rapidly differentiate the orthogonal probes in vivo will also be developed. Our approach is highly innovative, as it combines a unique blend of chemical, biological, and computational techniques to fill a long-standing void in imaging capabilities. The proposed research is significant, as the bioluminescent tools will enable the direct interrogation of cellular networks not currently possible with existing toolsets. Such studies will provide some of the first macroscopic images of tumor heterogeneity and may fundamentally change existing views on cancer progression. Additionally, similar to other imaging technologies, the bioluminescent probes will likely inspire new discoveries in a broad spectrum of fields.